Ventricular Standstill Following Intravenous Erythromycin and Borderline Hypokalemia

Ventricular standstill (VS) is a potentially fatal arrhythmia that is usually associated with syncope, if prolonged and is rarely asymptomatic[1]. Its mechanism involves either a lack of supraventricular impulse or an interruption in the transmission of these signals from the atria to the ventricles, resulting in a sudden loss of cardiac output[2]. Although rare, ventricular arrhythmias have been associated with intravenous (IV) erythromycin. However, to our knowledge, VS has not been reported following the administration of IV erythromycin. The Authors describe a rare case of asymptomatic VS and subsequent third-degree atrioventricular block, following the administration of IV erythromycin in a 49-year-old woman with borderline hypokalemia. Through this case, the Authors highlight the importance of cardiac monitoring and electrolyte replacement when administering IV erythromycin, as well as discuss several other mechanisms that contribute to ventricular arrhythmias.

The prevalence of genotypes that determine resistance to macrolides, lincosamides, and streptogramins B compared with spiramycin susceptibility among erythromycin-resistant Staphylococcus epidermidis

Coagulase-negative staphylococci, particularly Staphylococcus epidermidis , can be regarded as potential reservoirs of resistance genes for pathogenic strains, e.g., Staphylococcus aureus . The aim of this study was to assess the prevalence of different resistance phenotypes to macrolide, lincosamide, and streptogramins B (MLSB) antibiotics among erythromycin-resistant S. epidermidis, together with the evaluation of genes promoting the following different types of MLSB resistance: ermA, ermB, ermC, msrA, mphC, and linA/A’. Susceptibility to spiramycin was also examined. Among 75 erythromycin-resistant S. epidermidis isolates, the most frequent phenotypes were macrolides and streptogramins B (MSB) and constitutive MLSB (cMLSB). Moreover, all strains with the cMLSB phenotype and the majority of inducible MLSB (iMLSB) isolates were resistant to spiramycin, whereas strains with the MSB phenotype were sensitive to this antibiotic. The D-shape zone of inhibition around the clindamycin disc near the spiramycin disc was found for some spiramycin-resistant strains with the iMLSB phenotype, suggesting an induction of resistance to clindamycin by this 16-membered macrolide. The most frequently isolated gene was ermC, irrespective of the MLSB resistance phenotype, whereas the most often noted gene combination was ermC, mphC, linA/A’. The results obtained showed that the genes responsible for different mechanisms of MLSB resistance in S. epidermidis generally coexist, often without the phenotypic expression of each of them.

Ureaplasma parvum : understanding the complexities of intra-amniotic infection in an ovine model

The human Ureaplasma species are the most frequently isolated bacteria from the upper genital tract of pregnant women and can cause clinically asymptomatic, intra-uterine infections, which are difficult to treat with antimicrobials. Ureaplasma infection of the upper genital tract during pregnancy has been associated with numerous adverse outcomes including preterm birth, chorioamnionitis and neonatal respiratory diseases. The mechanisms by which ureaplasmas are able to chronically colonise the amniotic fluid and avoid eradication by (i) the host immune response and (ii) maternally-administered antimicrobials, remain virtually unexplored. To address this gap within the literature, this study investigated potential mechanisms by which ureaplasmas are able to cause chronic, intra-amniotic infections in an established ovine model. In this PhD program of research the effectiveness of standard, maternal erythromycin for the treatment of chronic, intra-amniotic ureaplasma infections was evaluated. At 55 days of gestation pregnant ewes received an intra-amniotic injection of either: a clinical Ureaplasma parvum serovar 3 isolate that was sensitive to macrolide antibiotics (n = 16); or 10B medium (n = 16). At 100 days of gestation, ewes were then randomised to receive either maternal erythromycin treatment (30 mg/kg/day for four days) or no treatment. Ureaplasmas were isolated from amniotic fluid, chorioamnion, umbilical cord and fetal lung specimens, which were collected at the time of preterm delivery of the fetus (125 days of gestation). Surprisingly, the numbers of ureaplasmas colonising the amniotic fluid and fetal tissues were not different between experimentally-infected animals that received erythromycin treatment or infected animals that did not receive treatment (p > 0.05), nor were there any differences in fetal inflammation and histological chorioamnionitis between these groups (p > 0.05). These data demonstrate the inability of maternal erythromycin to eradicate intra-uterine ureaplasma infections. Erythromycin was detected in the amniotic fluid of animals that received antimicrobial treatment (but not in those that did not receive treatment) by liquid chromatography-mass spectrometry; however, the concentrations were below therapeutic levels (<10 – 76 ng/mL). These findings indicate that the ineffectiveness of standard, maternal erythromycin treatment of intra-amniotic ureaplasma infections may be due to the poor placental transfer of this drug. Subsequently, the phenotypic and genotypic characteristics of ureaplasmas isolated from the amniotic fluid and chorioamnion of pregnant sheep after chronic, intra-amniotic infection and low-level exposure to erythromycin were investigated. At 55 days of gestation twelve pregnant ewes received an intra-amniotic injection of a clinical U. parvum serovar 3 isolate, which was sensitive to macrolide antibiotics. At 100 days of gestation, ewes received standard maternal erythromycin treatment (30 mg/kg/day for four days, n = 6) or saline (n = 6). Preterm fetuses were surgically delivered at 125 days of gestation and ureaplasmas were cultured from the amniotic fluid and the chorioamnion. The minimum inhibitory concentrations (MICs) of erythromycin, azithromycin and roxithromycin were determined for cultured ureaplasma isolates, and antimicrobial susceptibilities were different between ureaplasmas isolated from the amniotic fluid (MIC range = 0.08 – 1.0 mg/L) and chorioamnion (MIC range = 0.06 – 5.33 mg/L). However, the increased resistance to macrolide antibiotics observed in chorioamnion ureaplasma isolates occurred independently of exposure to erythromycin in vivo. Remarkably, domain V of the 23S ribosomal RNA gene (which is the target site of macrolide antimicrobials) of chorioamnion ureaplasmas demonstrated significant variability (125 polymorphisms out of 422 sequenced nucleotides, 29.6%) when compared to the amniotic fluid ureaplasma isolates and the inoculum strain. This sequence variability did not occur as a consequence of exposure to erythromycin, as the nucleotide substitutions were identical between chorioamnion ureaplasmas isolated from different animals, including those that did not receive erythromycin treatment. We propose that these mosaic-like 23S ribosomal RNA gene sequences may represent gene fragments transferred via horizontal gene transfer. The significant differences observed in (i) susceptibility to macrolide antimicrobials and (ii) 23S ribosomal RNA sequences of ureaplasmas isolated from the amniotic fluid and chorioamnion suggests that the anatomical site from which they were isolated may exert selective pressures that alter the socio-microbiological structure of the bacterial population, by selecting for genetic changes and altered antimicrobial susceptibility profiles. The final experiment for this PhD examined antigenic size variation of the multiple banded antigen (MBA, a surface-exposed lipoprotein and predicted ureaplasmal virulence factor) in chronic, intra-amniotic ureaplasma infections. Previously defined ‘virulent-derived’ and ‘avirulent-derived’ clonal U. parvum serovar 6 isolates (each expressing a single MBA protein) were injected into the amniotic fluid of pregnant ewes (n = 20) at 55 days of gestation, and amniotic fluid was collected by amniocentesis every two weeks until the time of near-term delivery of the fetus (at 140 days of gestation). Both the avirulent and virulent clonal ureaplasma strains generated MBA size variants (ranging in size from 32 – 170 kDa) within the amniotic fluid of pregnant ewes. The mean number of MBA size variants produced within the amniotic fluid was not different between the virulent (mean = 4.2 MBA variants) and avirulent (mean = 4.6 MBA variants) ureaplasma strains (p = 0.87). Intra-amniotic infection with the virulent strain was significantly associated with the presence of meconium-stained amniotic fluid (p = 0.01), which is an indicator of fetal distress in utero. However, the severity of histological chorioamnionitis was not different between the avirulent and virulent groups. We demonstrated that ureaplasmas were able to persist within the amniotic fluid of pregnant sheep for 85 days, despite the host mounting an innate and adaptive immune response. Pro-inflammatory cytokines (interleukin (IL)-1â, IL-6 and IL-8) were elevated within the chorioamnion tissue of pregnant sheep from both the avirulent and virulent treatment groups, and this was significantly associated with the production of anti-ureaplasma IgG antibodies within maternal sera (p < 0.05). These findings suggested that the inability of the host immune response to eradicate ureaplasmas from the amniotic cavity may be due to continual size variation of MBA surface-exposed epitopes. Taken together, these data confirm that ureaplasmas are able to cause long-term in utero infections in a sheep model, despite standard antimicrobial treatment and the development of a host immune response. The overall findings of this PhD project suggest that ureaplasmas are able to cause chronic, intra-amniotic infections due to (i) the limited placental transfer of erythromycin, which prevents the accumulation of therapeutic concentrations within the amniotic fluid; (ii) the ability of ureaplasmas to undergo rapid selection and genetic variation in vivo, resulting in ureaplasma isolates with variable MICs to macrolide antimicrobials colonising the amniotic fluid and chorioamnion; and (iii) antigenic size variation of the MBA, which may prevent eradication of ureaplasmas by the host immune response and account for differences in neonatal outcomes. The outcomes of this program of study have improved our understanding of the biology and pathogenesis of this highly adapted microorganism.

The epidemiology of invasive group A streptococcal disease in Victoria, Australia

Objective To estimate the incidence and severity of invasive group A streptococcal infection in Victoria, Australia. Design Prospective active surveillance study. Setting Public and private laboratories, hospitals and general practitioners throughout Victoria. Patients eople in Victoria diagnosed with group A streptococcal disease notified to the surveillance system between 1 March 2002 and 31 August 2004. Main outcome measure Confirmed invasive group A streptococcal disease. Results We identified 333 confirmed cases: an average annualised incidence rate of 2.7 (95% CI, 2.3-3.2) per 100000 population per year. Rates were highest in people aged 65 years and older and those younger than 5 years. The case-fatality rate was 7.8%. Streptococcal toxic shock syndrome occurred in 48 patients (14.4%), with a case-fatality rate of 23%. Thirty cases of necrotising fasciitis were reported; five (17%) of these patients died. Type 1 (23%) was the most frequently identified emm sequence type in all, age groups. All tested isolates were susceptible to penicillin and clindamycin. Two isolates (4%) were resistant to erythromycin. Conclusion The incidence of invasive group A streptococcal disease in temperate Australia is greater than previously appreciated and warrants greater public health attention, including its designation as a notifiable disease.

Ureaplasma parvum undergoes selection in utero resulting in genetically diverse isolates colonising the chorioamnion of fetal sheep

Ureaplasmas are the microorganisms most frequently isolated from the amniotic fluid of pregnant women and can cause chronic intrauterine infections. These tiny bacteria are thought to undergo rapid evolution and exhibit a hypermutatable phenotype; however, little is known about how ureaplasmas respond to selective pressures in utero. Using an ovine model of chronic intra-amniotic infection, we investigated if exposure of ureaplasmas to sub-inhibitory concentrations of erythromycin could induce phenotypic or genetic indicators of macrolide resistance. At 55 days gestation, 12 pregnant ewes received an intra-amniotic injection of a non-clonal, clinical U. parvum strain, followed by: (i) erythromycin treatment (IM, 30 mg/kg/day, n=6); or (ii) saline (IM, n=6) at 100 days gestation. Fetuses were then delivered surgically at 125 days gestation. Despite injecting the same inoculum into all ewes, significant differences between amniotic fluid and chorioamnion ureaplasmas were detected following chronic intra-amniotic infection. Numerous polymorphisms were observed in domain V of the 23S rRNA gene of ureaplasmas isolated from the chorioamnion (but not the amniotic fluid), resulting in a mosaic-like sequence. Chorioamnion isolates also harboured the macrolide resistance genes erm(B) and msr(D) and were associated with variable roxithromycin minimum inhibitory concentrations. Remarkably, this variability occurred independently of exposure of ureaplasmas to erythromycin, suggesting that low-level erythromycin exposure does not induce ureaplasmal macrolide resistance in utero. Rather, the significant differences observed between amniotic fluid and chorioamnion ureaplasmas suggest that different anatomical sites may select for ureaplasma sub-types within non-clonal, clinical strains. This may have implications for the treatment of intrauterine ureaplasma infections.

Crystallization as a Tool for Controlling and Designing Properties of Pharmaceutical Solids

The ability to deliver the drug to the patient in a safe, efficacious and cost-effective manner depends largely on the physicochemical properties of the active pharmaceutical ingredient (API) in the solid state. In this context, crystallization is of critical importance in pharmaceutical industry, as it defines physical and powder properties of crystalline APIs. An improved knowledge of the various aspects of crystallization process is therefore needed. The overall goal of this thesis was to gain better understanding of the relationships between crystallization, solid-state form and properties of pharmaceutical solids with a focus on a crystal engineering approach to design technological properties of APIs. Specifically, solid-state properties of the crystalline forms of the model APIs, erythromycin A and baclofen, and the influence of solvent on their crystallization behavior were investigated. In addition, the physical phenomena associated with wet granulation and hot-melting processing of the model APIs were examined at the molecular level. Finally, the effect of crystal habit modification of a model API on its tabletting properties was evaluated. The thesis enabled the understanding of the relationship between the crystalline forms of the model APIs, which is of practical importance for solid-state control during processing and storage. Moreover, a new crystalline form, baclofen monohydrate, was discovered and characterized. Upon polymorph screening, erythromycin A demonstrated high solvate-forming propensity thus emphasizing the need for careful control of the solvent effects during formulation. The solvent compositions that yield the desirable crystalline form of erythromycin A were defined. Furthermore, new examples on solvent-mediated phase transformations taking place during wet granulation of baclofen and hot-melt processing of erythromycin A dihydrate with PEG 6000 are reported. Since solvent-mediated phase transformations involve the crystallization of a stable phase and hence affect the dissolution kinetics and possibly absorption of the API these transformations must be well documented. Finally, a controlled-crystallization method utilizing HPMC as a crystal habit modifier was developed for erythromycin A dihydrate. The crystals with modified habit were shown to posses improved compaction properties as compared with those of unmodified crystals. This result supports the idea of morphological crystal engineering as a tool for designing technological properties of APIs and is of utmost practical interest.

Investigating physical properties of solid dosage forms during pharmaceutical processing : Process analytical applications of vibrational spectroscopy

In order to improve and continuously develop the quality of pharmaceutical products, the process analytical technology (PAT) framework has been adopted by the US Food and Drug Administration. One of the aims of PAT is to identify critical process parameters and their effect on the quality of the final product. Real time analysis of the process data enables better control of the processes to obtain a high quality product. The main purpose of this work was to monitor crucial pharmaceutical unit operations (from blending to coating) and to examine the effect of processing on solid-state transformations and physical properties. The tools used were near-infrared (NIR) and Raman spectroscopy combined with multivariate data analysis, as well as X-ray powder diffraction (XRPD) and terahertz pulsed imaging (TPI). To detect process-induced transformations in active pharmaceutical ingredients (APIs), samples were taken after blending, granulation, extrusion, spheronisation, and drying. These samples were monitored by XRPD, Raman, and NIR spectroscopy showing hydrate formation in the case of theophylline and nitrofurantoin. For erythromycin dihydrate formation of the isomorphic dehydrate was critical. Thus, the main focus was on the drying process. NIR spectroscopy was applied in-line during a fluid-bed drying process. Multivariate data analysis (principal component analysis) enabled detection of the dehydrate formation at temperatures above 45°C. Furthermore, a small-scale rotating plate device was tested to provide an insight into film coating. The process was monitored using NIR spectroscopy. A calibration model, using partial least squares regression, was set up and applied to data obtained by in-line NIR measurements of a coating drum process. The predicted coating thickness agreed with the measured coating thickness. For investigating the quality of film coatings TPI was used to create a 3-D image of a coated tablet. With this technique it was possible to determine coating layer thickness, distribution, reproducibility, and uniformity. In addition, it was possible to localise defects of either the coating or the tablet. It can be concluded from this work that the applied techniques increased the understanding of physico-chemical properties of drugs and drug products during and after processing. They additionally provided useful information to improve and verify the quality of pharmaceutical dosage forms

Antibiotic resistance in Campylobacter jejuni and Campylobacter coli isolated from poultry in the South-East Queensland region

Objectives: The aim of this study was to determine the antimicrobial resistance patterns of 125 Campylobacter jejuni and 27 Campylobacter coli isolates from 39 Queensland broiler farms. Methods: Two methods, a disc diffusion assay and an agar-based MIC assay, were used. The disc diffusion was performed and interpreted as previously described (Huysmans MB, Turnidge JD. Disc susceptibility testing for thermophilic campylobacters. Pathology 1997; 29: 209–16), whereas the MIC assay was performed according to CLSI (formerly NCCLS) methods and interpreted using DANMAP criteria. Results: In both assays, no C. jejuni or C. coli isolates were resistant to ciprofloxacin or chloramphenicol, no C. coli were resistant to nalidixic acid, and no C. jejuni were resistant to erythromycin. In the MIC assay, no C. jejuni isolate was resistant to nalidixic acid, whereas three isolates (2.4%) were resistant in the disc assay. The highest levels of resistance of the C. jejuni isolates were recorded for tetracycline (19.2% by MIC and 18.4% by disc) and ampicillin (19.2% by MIC and 17.6% by disc). The C. coli isolates gave very similar results (tetracycline resistance 14.8% by both MIC and disc; ampicillin resistance 7.4% by MIC and 14.8% by disc). Conclusions: This work has shown that the majority of C. jejuni and C. coli isolates were susceptible to the six antibiotics tested by both disc diffusion and MIC methods. Disc diffusion represents a suitable alternative methodology to agar-based MIC methods for poultry Campylobacter isolates.

Campylobacter Infections of Domestic Origin in Finland

Campylobacter jejuni and C. coli are the leading causes of human bacterial gastroenteritis in developed countries. Most human Campylobacter infections are sporadic and a seasonal peak in the distribution of infections can be seen in the summer months in several countries, including Finland. A variety of risk factors for Campylobacter infections have been identified; handling and eating poultry, drinking unpasteurized milk, contact with domestic animals, and travelling abroad. However, the relative importance of the different risk factors in sporadic cases of Campylobacter infection remains unknown. In most cases, the infection is self-limiting and no specific treatment is required. Campylobacter enteritis can cause a wide range of complications, including reactive arthritis (ReA) that is reported in 1-5% of the cases. Seven clinical microbiology laboratories serving different geographical areas of Finland, participated in this multi-centre study, conducted during a seasonal peak in 2002. In a matched case-control study, domestically-acquired sporadic Campylobacter infections from three geographical areas were collected. The final study comprised 100 cases and 137 controls. Risk factors for sporadic domestically-acquired Campylobacter infections were identified on the basis of a questionnaire; swimming in natural waters was found to be a novel risk factor for Campylobacter infection. Other independent risk factors were tasting or eating raw or undercooked meat and drinking untreated water from a dug well. The role of bacterial strain and host characteristics are not fully understood in Campylobacter infections. Exposure factors, demographical characteristics, and the serotype of the Campylobacter isolate may affect the severity of the enteritis. This cross-sectional study comprised 114 patients with C. jejuni enteritis, diagnosed in three clinical microbiology laboratories; most of the patients had participated in the previous case-control study. Swimming was associated with age ≤ 5 years and serotype Pen 6,7 was found significantly more often among patients reporting swimming. The geographical distribution among serotypes varied; serotype Pen 4-complex appeared more often in patients from urban areas and serotype Pen 21 among patients from more rural areas. Thus, risk factors and sources of infection for C. jejuni infection may vary among individuals depending on age and geographical location. The in vitro susceptibilities of C. jejuni and C. coli strains isolated from patients infected abroad (85 strains) or domestically (393 strains) revealed that susceptibility to erythromycin is still high, even among isolates of foreign origin. However, the novel antimicrobial agent telithromycin did not offer any advantage over erythromycin; isolates with high minimal inhibitory concentrations (MICs) for erythromycin also showed reduced susceptibility to telithromycin. Reduced susceptibility to fluoroquinolones was detected almost exclusively among isolates of foreign origin and half of these isolates with high MICs for fluoroquinolones also showed elevated MICs for doxycycline. Questionnaires concerning complications associated with C. jejuni enteritis were sent to patients two months after becoming ill; 201 patients from seven different geographical areas were included in the study. Musculoskeletal complications after C. jejuni infection were commonly reported by patients (39%). The incidence of classical ReA was 4% and that of Achilles enthesopathy and/or heel pain 9%. Other C. jejuni-associated reactive joint symptoms were commonly reported, however, due to their milder nature seldom seen and diagnosed by a physician. The severity of the enteritis may predict further complications; stomach ache during enteritis was connected to the development of later joint pain. Early antimicrobial treatment, within two days from the start of symptoms, shortened the duration of diarrhoea by two days but did not prevent later musculoskeletal complications. Campylobacter is an important human enteropathogen and causes a significant burden of illness. As the incidence of Campylobacter infections is high, the importance of the infection and the occurrence of complications will increase. This stresses the importance of understanding the risk factors for acquiring Campylobacter infection and how bacterial strain and host characteristics may affect the risk for infection. The role of antimicrobial treatment for acute Campylobacter enteritis seems to be marginal and should be used restrictively.

Use of a proposed antimicrobial susceptibility testing method for Haemophilus parasuis

The aim of this study was to examine the antimicrobial susceptibility of 97 Haemophilus parasuis cultured from Australian pigs. As there is no existing standard antimicrobial susceptibility technique available for H. parasuis, methods utilising the supplemented media, BA/SN for disc diffusion and test medium broth (TMB) for a microdilution technique, were initially evaluated with the reference strains recommended by the Clinical and Laboratory Standards Institute. The results of the media evaluation suggested that BA/SN and TMB can be used as suitable media for susceptibility testing of H. parasuis. The proposed microdilution technique was then used with 97 H. parasuis isolates and nine antimicrobial agents. The study found that Australian isolates showed elevated minimum inhibitory concentrations (MICs) for ampicillin (1%), penicillin (2%), erythromycin (7%), tulathromycin (9%), tilmicosin (22%), tetracycline (31%) and trimethoprim-sulfamethoxazole (40%). This study has described potential antimicrobial susceptibility methods for H. parasuis and has detected a low percentage of Australian H. parasuis isolates with elevated antimicrobial MICs.

Cytochrome P450-mediated drug interactions affecting lidocaine

Lidocaine is a widely used local anaesthetic agent that also has anti-arrhythmic effects. It is classified as a type Ib anti-arrhythmic agent and is used to treat ventricular tachycardia or ventricular fibrillation. Lidocaine is eliminated mainly by metabolism, and less than 5% is excreted unchanged in urine. Lidocaine is a drug with a medium to high extraction ratio, and its bioavailability is about 30%. Based on in vitro studies, the earlier understanding was that CYP3A4 is the major cytochrome P450 (CYP) enzyme involved in the metabolism of lidocaine. When this work was initiated, there was little human data on the effect of inhibitors of CYP enzymes on the pharmacokinetics of lidocaine. Because lidocaine has a low therapeutic index, medications that significantly inhibit lidocaine clearance (CL) could increase the risk of toxicity. These studies investigated the effects of some clinically important CYP1A2 and CYP3A4 inhibitors on the pharmacokinetics of lidocaine administered by different routes. All of the studies were randomized, double-blind, placebo-controlled cross-over studies in two or three phases in healthy volunteers. Pretreatment with clinically relevant doses of CYP3A4 inhibitors erythromycin and itraconazole or CYP1A2 inhibitors fluvoxamine and ciprofloxacin was followed by a single dose of lidocaine. Blood samples were collected to determine the pharmacokinetic parameters of lidocaine and its main metabolites monoethylglycinexylidide (MEGX) and 3-hydroxylidocaine (3-OH-lidocaine). Itraconazole and erythromycin had virtually no effect on the pharmacokinetics of intravenous lidocaine, but erythromycin slightly prolonged the elimination half-life (t½) of lidocaine (Study I). When lidocaine was taken orally, both erythromycin and itraconazole increased the peak concentration (Cmax) and the area under the concentration-time curve (AUC) of lidocaine by 40-70% (Study II). Compared with placebo and itraconazole, erythromycin increased the Cmax and the AUC of MEGX by 40-70% when lidocaine was given intravenously or orally (Studies I and II). The pharmacokinetics of inhaled lidocaine was unaffected by concomitant administration of itraconazole (Study III). Fluvoxamine reduced the CL of intravenous lidocaine by 41% and prolonged the t½ of lidocaine by 35%. The mean AUC of lidocaine increased 1.7-fold (Study IV). After oral administration of lidocaine, the mean AUC of lidocaine in-creased 3-fold and the Cmax 2.2-fold by fluvoxamine (Study V). During the pretreatment with fluvoxamine combined with erythromycin, the CL of intravenous lidocaine was 53% smaller than during placebo and 21% smaller than during fluvoxamine alone. The t½ of lidocaine was significantly longer during the combination phase than during the placebo or fluvoxamine phase. The mean AUC of intravenous lidocaine increased 2.3-fold and the Cmax 1.4-fold (Study IV). After oral administration of lidocaine, the mean AUC of lidocaine increased 3.6-fold and the Cmax 2.5-fold by concomitant fluvoxamine and erythromycin. The t½ of oral lidocaine was significantly longer during the combination phase than during the placebo (Study V). When lidocaine was given intravenously, the combination of fluvoxamine and erythromycin prolonged the t½ of MEGX by 59% (Study IV). Compared with placebo, ciprofloxacin increased the mean Cmax and AUC of intravenous lidocaine by 12% and 26%, respectively. The mean plasma CL of lidocaine was reduced by 22% and its t½ prolonged by 7% (Study VI). These studies clarify the principal role of CYP1A2 and suggest only a modest role of CYP3A4 in the elimination of lidocaine in vivo. The inhibition of CYP1A2 by fluvoxamine considerably reduces the elimination of lidocaine. Concomitant use of fluvoxamine and the CYP3A4 inhibitor erythromycin further increases lidocaine concentrations. The clinical implication of this work is that clinicians should be aware of the potentially increased toxicity of lidocaine when used together with inhibitors of CYP1A2 and particularly with the combination of drugs inhibiting both CYP1A2 and CYP3A4 enzymes.

Lactobacillus bacteremia, with special focus on the safety of probiotic Lactobacillus rhamnosus GG

Lactobacilli are gram positive rods, which belong to normal oropharyngeal, gastrointestinal and urogenital flora. They are widely used in food industry and as food additives. Although their virulence is presumed to be very low, opportunistic bacteremic infections, especially in immunocompromised hosts, have been detected. In the present study, the possible effects of increasing probiotic use of Lactobacillus rhamnosus GG (LGG) on the occurrence of bacteremia due to lactobacilli was evaluated on population level. In Finland, 90 Lactobacillus bacteremia cases were reported to the National Infectious Disease Register maintained by National Public Health Institute, during 1995-2000. Their proportion of all bacteremia cases was on average 0.24%, corresponding to 0.3 cases/100 000 inhabitants annually. In the Helsinki University Central Hospital district the corresponding proportion of all bacteremia cases was observed during 1990-2000. Despite LGG intake increased six folded no increasing trend in the occurrence of lactobacilli bacteremia was seen. A total of 85 Lactobacillus sp. blood isolates collected from different human bacteremic cases were characterised and compared with the commercial probiotic LGG strain. In species characterisation 46 L. rhamnosus strains, 12 L. fermentum and L. casei strains each, three each of L. gasseri, L. salivarius and L. jensenii species, two L. curvatus, and one each of L. plantarum, L. sakei, L. zeae and L. reuteri species were detected. Nearly half of the L. rhamnosus findings turned out to be indistinguishable from the probiotic LGG strain. Common predisposing factors to Lactobacillus bacteremia were immunosuppression, prior prolonged hospitalisation and prior surgical interventions. Severe or fatal comorbidities were found in 82% of the patients. Mortality at one month was 26% and severe underlying diseases were a significant predictor of death (OR 15.8). Antimicrobial susceptibility of Lactobacillus strains was species dependent. The Lactobacillus isolates were generally susceptible to imipenem, piperacillin-tazobactam, clindamycin and erythromycin, whereas all other than L. gasseri and L. jensenii species were not at all susceptible to vancomycin. The susceptibility to cephalosporin varied greatly even within species why they might not be recommended for treatment of Lactobacillus infections. The effect and safety of probiotic LGG preparation in amelioration of gastric symptoms and diarrhea in HIV-infected patients was evaluated. No significant differences in gastrointestinal symptoms or diarrhoea in LGG treated patients compared to placebo could be found. LGG was well tolerated with no adverse effects including bacteremic outbreaks could be observed. The use of probiotic LGG can be regarded safe in this immunocompromised patient group.

Finnish cattle as reservoir of Campylobacter spp.

The reported incidence of human campylobacteriosis in Finland is higher than in most other European countries. A high annual percentage of sporadic infections is of foreign origin, although a notable proportion of summer infections is domestically acquired. While chickens appear to be a major source of campylobacters for humans in most countries, the prevalence of campylobacters is very low in chicken slaughter batches in Finland. Data on other potential animal reservoirs of human pathogenic campylobacters in Finland are scarce. Consequently, this study aimed to investigate the status of Finnish cattle as a potential source of thermophilic Campylobacter spp. and antibiotic-resistant Campylobacter jejuni for human sporadic campylobacter infections of domestic origin. A survey of the prevalence of thermophilic Campylobacter spp. in Finnish cattle studied bovine rectal faecal samples (n=952) and carcass surface samples (n=948) from twelve Finnish slaughterhouses from January to December 2003. The total prevalence of Campylobacter spp. in faecal samples was 31.1%, and in carcass samples 3.5%. Campylobacter jejuni, the most common species, was present in 19.5% of faecal samples and in 3.1% of carcasses. In addition to thermophilic Campylobacter spp., C. hyointestinalis ssp. hyointestinalis was present in bovine samples. The prevalence of campylobacters was higher among beef cattle than among dairy cattle. Using the enrichment method, the number of positive faecal samples was 7.5 times higher than that obtained by direct plating. The predominant serotypes of faecal C. jejuni, determined by serotyping with a set of 25 commercial antisera for heat-stable antigens (Penner), were Pen2 and Pen4-complex, which covered 52% of the samples. Genotyping with pulsed-field gel electrophoresis (PFGE) using SmaI restriction yielded a high diversity of C. jejuni subtypes in cattle. Determining the minimum inhibitory concentrations of ampicillin, enrofloxacin, erythromycin, gentamicin, nalidixic acid, and oxytetracycline among bovine C. jejuni isolates using a commercial broth microdilution method yielded 9% of isolates resistant to at least one of the antimicrobials examined. No multiresistant isolates were found among the bovine C. jejuni strains. The study of the shedding patterns of Campylobacter spp. among three Finnish dairy cattle herds included the examination of fresh faecal samples and tank milk samples taken five times, as well as samples from drinking troughs taken once during the one-year study. The semiquantitative enrichment method detected C. jejuni in 169 of the 340 faecal samples, mostly at low levels. In addition, C. jejuni was present in one drinking trough sample. The prevalence between herds and sampling occasions varied widely. PFGE, using SmaI as restriction enzyme, identified only a few subtypes in each herd. In two 2 of the herds, two subtypes persisted throughout the sampling. Individual animals presented various shedding patterns during the study. Comparison of C. jejuni isolates from humans, chickens and cattle included the design of primers for four new genetic markers selected from completely sequenced C. jejuni genomes 81-176, RM1221 and NCTC 11168, and the PCR examination of domestic human isolates from southern Finland in 1996, 2002 and 2003 (n=309), chicken isolates from 2003, 2006 and 2007 (n=205), and bovine isolates from 2003 (n=131). The results revealed that bovine isolates differed significantly from human and chicken isolates. In particular, the - glutamyl transpeptidase gene was uncommon among bovine isolates. The PFGE genotyping of C. jejuni isolates, using SmaI and KpnI restriction enzymes, included a geographically representative collection of isolates from domestic sporadic human infections, chicken slaughter batches, and cattle faeces and carcasses during the seasonal peak of campylobacteriosis in the summer of 2003. The study determined that 55.4% of human isolates were indistinguishable from those of chickens and cattle. Temporal association between isolates from humans and chickens was possible in 31.4% of human infections. Approximately 19% of the human infections may have been associated with cattle. However, isolates from bovine carcasses and human cases represented different PFGE subtypes. In conclusion, this study suggests that Finnish cattle is a notable reservoir of C. jejuni, the most important Campylobacter sp. in human enteric infections. Although the concentration of these organisms in bovine faeces appeared to be low, excretion can be persistent. The genetic diversity and presence or absence of marker genes support previous suggestions of host-adapted C. jejuni strains, and may indicate variations in virulence between strains from different hosts. In addition to chickens, Finnish cattle appeared to be an important reservoir and possible source of C. jejuni in domestic sporadic human infections. However, sources of campylobacters may differ between rural and urban areas in Finland, and in general, the transmission of C. jejuni of bovine origin probably occurs via other routes than food.

Influência de pré-tratamentos de células epiteliais com penicilina e eritromicina na aderência e na viabilidade intracelular de Corynebacterium diphtheriae

A difteria é uma síndrome toxêmica causada pelo Corynebacterium diphtheriae. Embora programas de imunização mantenham a doença sob controle em países desenvolvidos, a difteria ainda permanece endêmica em diversas partes do mundo, especialmente em indivíduos parcialmente imunizados para toxina diftérica. Junto a isso, nos últimos 20 anos, tem-se observado a ocorrência crescente de quadros de infecções sistêmicas causados por cepas atoxinogênicas. A penicilina e eritromicina são as principais drogas de escolha no tratamento destas infecções, entretanto, a escassez de trabalhos reavaliando a terapia de escolha e a influência dos antibióticos no processo de interação bacteriana com células epiteliais humanas são escassos, logo compreendem aspectos que justificam o presente estudo. O objetivo principal deste projeto consiste na análise da influência do pré-tratamento de células epiteliais Hep-2 com os antimicrobianos penicilina e eritromicina na colonização e viabilidade intracelular de amostras de C. diphtheriae. As monocamadas foram submetidas ao tratamento prévio com doses séricas terapêuticas de penicilina (5g mL1) e eritromicina (1,92 g mL1) por 24 horas e os antibióticos foram removidos por lavagens com PBS antes da interação com a suspensão bacteriana (MOI de 107). Três horas após a infecção, o padrão de aderência foi investigado como também, realizada a análise das bactérias viáveis associadas e internalizadas nas monocamadas. O pré-tratamento com penicilina induziu a formação de perfil de aderência difuso (AD) pelas amostras estudadas. Microorganismos que normalmente apresentam padrão de aderência localizado (AL) passaram a expressar perfil (AD) após pré-tratamento das camadas com ambos antimicrobianos. A expressão do tipo agregativo (AA) não foi influenciada pela presença de eritromicina. A penicilina e a eritromicina reduziram o número de bactérias viáveis associadas às células HEp-2 na maioria das oportunidades. Entretanto, a penicilina interferiu em maior magnitude nesse processo. As três amostras invasoras de C. diphtheriae (HC01, HC04 e BR5015), apresentaram maior capacidade de sobrevivência no compartimento intracelular, independente do pré-tratamento. A expressão dos perfis de aderência assim como a capacidade de sobrevivência no compartimento intracelular frente aos antimicrobianos testados mostraram-se independentes da produção de toxina e dos percentuais de associação com as células HEp-2. Foi observada uma maior eficiência da eritromicina na eliminação de bactérias viáveis internalizadas reforçando a utilização clínica da eritromicina tanto no tratamento de pacientes quanto na erradicação do estado de portador. Novos estudos serão desenvolvidos para investigar alterações na expressão de fatores de virulência por amostras de C. diphtheriae na interação com células HEp-2 pré-tratadas com antimicrobianos e a influência sobre a evolução clínica das infecções por corinebactérias

Perfil genético de Enteroccocus faecalis isolados de infecção endodôntica primária no Brasil comparados a isolados orais e não-orais do Reino Unido e do Japão

Enterococcus faecalis (E. faecalis), conhecidamente patógeno oportunista, tem sido frequentemente associado a infecções sistêmicas graves. É também encontrado na cavidade oral, com destaque em infecção endodôntica refratária. O objetivo deste estudo foi avaliar características moleculares de E. faecalis isolados de infecção endodôntica primária no Brasil e comparar com isolados orais e não orais de pacientes do Reino Unido e do Japão, assim como E. faecalis resistentes à vancomicina. O presente estudo também investigou o relacionamento entre E. faecalis de diferentes origens (oral e não oral) e de diferentes áreas geográficas para obter uma melhor compreensão do envolvimento dos diferentes reservatórios no surgimento e propagação de clones virulentos, aqueles que possuem genes que conferem infectividade e virulência, assim como resistência aos antibióticos. Para tal, foram estudados E. faecalis isolados em infecções endodônticas no Brasil (n = 20) e orais no Reino Unido (n = 10), e em infecções não orais no Japão (n = 9). Além disso, 20 E. faecalis isolados ambientais do Hospital Universitário de Gales (Cardiff, Reino Unido), classificados como Enterococcus resistentes à vancomicina (VRE) também foram examinados. A Concentração Inibitória Mínima (CIM) dos isolados do Brasil foi obtida pelo método de diluição em agar de acordo com as recomendações do Clinical and Laboratory Standards Institute (CLSI). Reação em cadeia da polimerase (inglês - PCR) foi a técnica empregada para detectar os genes de virulência e aqueles associados à resistência aos antibióticos, enquanto Reação de Amplificação Aleatória de DNA Polimórfico (inglês - RAPD-PCR) foi escolhida para a tipagem molecular. Dentre os genes de virulência examinados, o gene que codifica a gelatinase gelE foi o mais prevalente entre os isolados (77-100%). Entre isolados orais, foram detectados os genes agg de substâncias de agregação, esp de proteína de evasão imune, cylB de citolisina, genes de resistência à tetraciclina tetM e tetL e à eritromicina ermB com diferentes prevalências. Os isolados clínicos hospitalares do Japão apresentaram perfil genético similar aos isolados orais, mas com maior prevalência de ermB e cylB. Todas as amostras de VRE foram positivas para os genes gelE, esp, agg, vanA, ermB e tetM, 95% foram positivos para cylB e 17% positivo para tetL. Todas as amostras foram negativas para ermA, asa373, vanB, vanC1 e vanC2/3. RAPD-PCR revelou agrupamento de VRE em comparação com outros isolados. Neste estudo, os isolados de E. faecalis de infecções orais apresentaram genes de resistência à tetraciclina, um antimicrobiano frequentemente usado no tratamento local de infecções dentárias, abrindo um debate muito importante sobre o papel e a eficácia desta droga para infecções orais. Claramente, são necessários mais estudos nesta área principalmente em relação à expressão de fatores de virulência entre isolados endodônticos para melhor nortear as estratégias de tratamento. As pressões externas no microambiente dos canais radiculares podem ser responsáveis pela seleção de espécies mais resistentes e virulentas. Por fim, embora isolados orais apresentem genes de virulência fundamentais para a patogenicidade, estes foram detectados em menor incidência em comparação com os isolados não-orais e VRE.